Rotation of treatments between spinosad and amitraz for the control of Rhipicephalus (Boophilus) microplus populations with amitraz resistance.

A farmlet study was conducted over 4 years in which three treatments were applied to six groups of Holstein dairy calves. Calves in each group were infested with equal numbers of N-strain (susceptible) and Ultimo strain (amitraz and synthetic pyrethroid resistant) tick larvae to establish self-sustaining populations with an initial, measurable level of resistance to amitraz. Standard counts of all ticks between 4.5 and 8.0mm diameter on one side of each animal were made each week and treatment was applied when tick numbers exceeded a threshold of 25 engorged adults per side. The three treatments were: 1, spinosad spray whenever tick numbers exceeded the threshold; 2, amitraz spray whenever tick numbers exceeded the threshold; 3, spinosad whenever tick numbers exceeded the threshold for the first 2 months, then amitraz for 2 months, with alternation every subsequent 2 months. Engorged adult female ticks were collected from each treatment group on 10 or 11 occasions during the study and tested using the larval packet test bioassay (LPT) for acaricide resistance. Spinosad 250ppm provided effective control of amitraz-resistant tick populations in the field, using a similar number of treatments as in the amitraz and rotation groups. The initial infestations of all of the groups resulted in the establishment of populations with in vitro evidence of resistance to amitraz using the LPT. Treatment with spinosad or with a rotation between spinosad and amitraz every 2 months resulted in reduced levels of resistance to amitraz according to the LPT. The animals treated with amitraz alone showed increasing resistance to amitraz according to the LPT each summer and autumn with a return to full or almost full susceptibility to amitraz in early spring in all years. This pattern suggests a relative lack of fitness of amitraz-resistant ticks that might be exploited by using an acaricide rotation strategy.

Toxicity of Bacillus thuringiensis to parasitic and free-living life-stages of nematode parasites of livestock.

A collection of Bacillus thuringiensis (Bt) strains (Bts) were screened for activity against the free-living larval stages of nematode parasites of livestock. Two strains were identified with significant activity in inhibiting larval development of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta. These strains were also toxic to the adult parasitic stages of these nematode species in vitro. Adult H. contortus and O. circumcincta showed complete cessation of movement within 2 and 4 days, respectively. Trichostrongylus colubriformis adults were less affected, however, movement was still significantly reduced compared with controls. The in vitro activity against the larval stages was of a magnitude similar to or greater than that seen with the anthelmintic drugs thiabendazole and levamisole. N-terminal amino acid sequencing indicated that the two Bts contained either Cry5A and Cry5B proteins, or a Cry13 protein, and the presence of the corresponding cry5A, cry5B and cry13 genes was confirmed by PCR and sequencing. Bacillus thuringiensis spore-crystal suspensions exposed to acidic pH conditions (pH

Comparison of 3 tests to detect acaricide resistance in Boophilus decoloratus on dairy farms in the Eastern Cape Province, South Africa.

The susceptibility of the larval offspring of engorged female Boophilus decoloratus, and of the engorged females, collected from cattle on the dairy farms Brycedale, Sunny Grove and Welgevind in the Eastern Cape Province, South Africa, was tested against the acaricides amitraz, chlorfenvinphos and cypermethrin. Resistance was determined by means of the Shaw Larval Immersion Test (SLIT) for larvae and the Reproductive Estimate Test (RET) and Egg Laying Test (ELT) for adults. At Brycedale the tests all indicated resistance to chlorfenvinphos, and RET and ELT indicated resistance to amitraz and emerging resistance to cypermethrin. At Sunny Grove, B. decoloratus was resistant to cypermethrin using SLIT and exhibited emerging resistance to chlorfenvinphos with SLIT and to cypermethrin with both RET and ELT At Welgevind, resistance was recorded against chlorfenvinphos (SLIT) and against cypermethrin (ELT), and emerging resistance against permethrin (RET). The results obtained with RET and ELT were generally comparable, but often differed from those obtained with SLIT. Resistance could be detected within 7 days with ELT compared to 42 days with RET and 60 days with SLIT.

Molecular cloning and expression of the dihydrofolate reductase (DHFR) gene from adult buffalo fly (Haematobia irritans exigua): effects of antifolates.

The folate analogues methotrexate, aminopterin and pyrimethamine were toxic when fed in a blood meal to adult buffalo flies (Haematobia irritans exigua), but aminopterin caused greater mortality than methotrexate, while trimethoprim was not toxic to adult flies. This is the first recorded instance of mortality in adult insects caused by ingestion of folate analogues. In order to investigate the mechanism of this toxicity, the dihydrofolate reductase (DHFR) gene was cloned from adult buffalo fly cDNA using a PCR-based approach. The full-length DHFR coding sequence (BF-DHFR) was 887 bp and contained an open reading frame encoding a protein of 188 amino acids. The deduced protein sequence identities between BF-DHFR and the other known insect DHFR sequences were: Drosophila melanogaster, 75%; Aedes albopictus, 54%; Heliothis virescens, 43%. The BF-DHFR gene has a single 52 bp intron, an organization more similar to Dipteran species (Drosophila and Aedes). The cDNA encoding BF-DHFR was inserted into an Escherichia coli expression vector and the recombinant protein was expressed to levels representing about 25% of total cell protein. The active enzyme was purified by affinity chromatography on methotrexate-agarose and displayed a relatively low affinity (IC50 = 30 nm) for methotrexate.

Tests to determine LC50 and discriminating doses for macrocyclic lactones against the cattle tick, Boophilus microplus.

Laboratory tests were carried out on larvae and adults of the cattle tick Boophilus microplus to determine the toxicity of macrocyclic lactone acaricides (MLs). Technical and commercial MLs were used in larval packet test (LPT), larval immersion test (LIT) and adult immersion test (AIT). In LIT and AIT the toxicity of MLs was much higher than for LPT. In the AIT, diluting the injectable formulation of MLs in water was as effective as dilution in ethanol+Triton X-100. LC50, LC99.9 and 95% confidence limits were determined so that a discriminating dose (DD) could be set for larval and adult tests in order to diagnose potential resistance to MLs in field samples of the tick. These DDs are for Australian strains of B. microplus and may not be suitable for other strains until further work is carried out. The value of these diagnostic tests can only be verified if or when resistance to MLs emerges in ticks.

Development of tick gut forms of Babesia bigemina in vitro.

Development of a laboratory cultured tick-transmissible strain of Babesia bigemina was followed in vitro after addition of gut material from engorged female Boophilus microplus ticks and incubation at 37 degrees C. Sequential development of stages, from intraerythrocytic strahlenkörper through multiplication to the fusion of what is assumed to be two gametes, is described. A change in physical environment (temperature, gas composition) experienced during passage of Babesia stages into the in vitro culture tubes possibly mimics the changes experienced in passage from host blood to the midgut of the tick vector. The effect in vitro was to induce the erythrocytic parasites to remain inactive at a trophozoite-like stage. Addition of factor(s) within midgut initiated further development of strahlenkörper. Two populations of strahlenkörper were recognized; an elongated form which did not appear to develop further, and a polymorphic population which underwent further multiplication initiated while the parasites were still within the erythrocyte, and continuing after they had emerged. These strahlenkörper increased in size as multiple division of nuclei occurred. with cell division being completed more slowly. Large aggregations of multinucleated strahlenkorper formed, but once division was complete, single-nucleated strahlenkörper emerged from the aggregates. Two individuals of post-aggregation strahlenkörper, assumed to be gametes, fused together. The morphology and ultrastructure of all stages of development are described and compared with forms already described from the tick midgut.

Establishment of Boophilus microplus infected with Babesia bigemina by using in vitro tube feeding technique.

The in vitro tube feeding technique is used to establish a laboratory colony of Boophilus microplus infected with Babesia bigemina. Pre-fed engorged female ticks offered 2 x 10(4) and 2 x 10(5)/ml of B. bigemina infected bovine red blood cells (RBC) showed sporokinetes in the haemolymph smear sample, and positive signals for B. bigemina in polymerase chain reaction (PCR). Larvae laid from the ticks offered 2 x 10(5)/ml of B. bigemina infected RBC showed evidence for B. bigemina infection in microscopic method and PCR. While larvae laid from the ticks offered 2 x 10(4)/ml of B. bigemina infected RBC showed positive for B. bigemina in only PCR. The females offered 2 x 10(3)/ml B. bigemina infected RBC and their larvae did not show positive evidences for B. bigemina infection. It is thought that the in vitro tube feeding technique can be a convenient method to study the relationship between ticks and tick-borne pathogens. It is also suggested that the superior sensitivity of PCR compared to the microscopic method in detection of B. bigemina from the tick sample, especially in larvae.

Digestion of host immunoglobulin and activity of midgut proteases in the buffalo fly Haematobia irritans exigua.

The digestion of blood by the buffalo fly (Haematobia irritans exigua) was monitored for 6h at 33 degrees C after a single meal. Following the meal, the concentration of soluble protein within the midgut increased to a peak at 2 hours then decreased steadily over the next 4h. The magnitude of the increase in soluble protein at 2h indicated a release of protein from another source; most likely from lysed red blood cells. The immunoglobulin (IgG) fraction of the blood meal was digested rapidly (50% within one hour of feeding) and fully digested within 4h. This is indicative of its accessibility to digestive enzymes within the midgut. In contrast, when flies had continuous access to blood, the concentration of IgG in the midgut remained at a more constant level. The loss of antigen-binding activity of a specific antibody was more rapid than complete degradation of the IgG, with 70% of binding activity lost within one hour of feeding. The level of trypsin activity in the midgut increased from pre-feeding levels to reach a peak at 2h before returning to basal levels after 6h. The pattern of trypsin activity follows closely that of the concentration of soluble protein in the midgut (r=0.88). The activity of leucine aminopeptidase in the midgut also increased immediately after feeding and remained elevated for 4 h before declining to a basal level after 6h. The rapid digestion of IgG and subsequent loss of antibody activity suggests that for a specific anti-buffalo fly antibody to be effective it would need to be able to either evade the digestive system or induce a rapid response.

Acid phosphatase in midgut digestive cells in partially fed females of the cattle tick Boophilus microplus.

Enzyme cytochemistry was used to identify vesicles containing acid phosphatase in the midgut digestive cells of partially fed females of the cattle tick Boophilus microplus. The vesicles were elongated or tubular in shape and appeared to be involved with the digestion of host bloodmeal. In mature cells, they were sometimes in close contact with large endosomes, which contained host blood. The vesicles were identified as tubular lysosomes because their morphological and cytochemical characteristics were analogous to similar structures described in mammalian cells. This is the first report of such lysosomes in tick gut cells and suggests some parallels with the intracellular structures involved in the digestion process of mammalian cells. Acid phosphatase in tick gut was also assayed biochemically and was shown to be inhibited with 10 mM sodium fluoride. Cytochemistry showed that this inhibitor blocked activity within the cell and on the lumenal cell membrane.

Comparison of prostaglandin E2 (PGE2) in salivary gland of Boophilus microplus, Haemaphysalis longicornis and Ixodes holocyclus, and quantification of PGE2 in saliva, hemolymph, ovary and gut of B. microplus.

The amount of prostaglandin E2 (PGE2) in salivary gland of semi-engorged adult female Boophilus microplus, Haemaphysalis longicornis and Ixodes holocyclus were 374.3 pg, 427.0 pg and 825.0 pg per one tick, respectively. It was thought that the PGE2 production is a common phenomenon among ticks. Then PGE2 concentrations in saliva and hemolymph, salivary gland, ovary and gut of fully-engorged adult female B. microplus were compared. The PGE2 concentration in saliva induced by pilocarpine was 40.3 ng/ml and hemolymph was 19.1 ng/ml. Salivary gland, ovary and gut from a tick contains 35.5 pg., 27.0 pg and 2.5 ng of PGE2, respectively.

Insecticides and acaricides: resistance and environmental impact.

Insecticides continue to be the primary means of control for ectoparasites on livestock. Intensive use of these materials has led to resistance to organochlorines, organophosphates and pyrethroids among populations of Haematobia irritans irritans, H. irritans exigua and Lucilia cuprina. Similarly, use of acaricides has led to resistance in one-host Boophilus ticks to all currently-used organophosphate-carbamates, synthetic pyrethroids and amidines. Resistance in multi-host ticks is less widespread. New chemicals are available for the control of resistant ectoparasites, but there are concerns over resistance and residues problems, which prompt the authors to discuss new pest management strategies. Environmental concerns are raised regarding the use of pesticides on livestock.

Prostaglandin E2 production by the cattle tick (Boophilus microplus) into feeding sites and its effect on the response of bovine mononuclear cells to mitogen.

Prostaglandin E2 (PGE2) secretion by the cattle tick Boophilus microplus into feeding sites was quantified. It was detected by the in vitro tube feeding experiment and it was determined that a semi-engorged female tick could produce and transmit 1.8 ng PGE2 into the feeding site. Using the in vitro membrane feeding system, newly molted adult ticks were also shown to secrete 0.04-0.15 ng PGE2 into the feeding site; however, female ticks produced more PGE2 than male ticks. The immune suppressive effect of PGE2 in the saliva of B. microplus on the bovine mononuclear cells (MNC) was also examined. PGE2 in the saliva was suspected of being a major component that inhibited the blastogenic response of MNC to a T-cell mitogen phytohemagglutinin. As bovine MNC are sensitive to low level concentration of PGE2, the PGE2 transmitted into feeding sites was suspected to be sufficient to produce physiological effects on the bovine host.

Generic approaches to obtaining efficacious antigens from vector arthropods.

The development of vaccines to control ectoparasites is dependent upon the identification of key parasite antigens. While a rational, pragmatic approach to antigen identification has yielded a successful vaccine candidate from ticks, there may be problems with such an approach when dealing with other ectoparasites. As an alternative approach, the search for vaccine candidates may be facilitated by cloning and expressing parasite genes encoding proteins involved in key physiological roles. A number of criteria may be applied to short-list candidate vaccines, these being; (a) host antibodies should be able to gain access to the parasite antigen; (b) sufficient antibody must gain access to the antigen target; (c) the formation of antibody-antigen complex should disrupt the normal function of the parasite antigen (d) the antigen should share conserved structural/sequence motifs with related, characterised, proteins, thus allowing the use of recombinant DNA methods to clone and express the candidate antigen. We propose three major groups of parasite antigens which may fulfill these criteria; serine proteases, chemoreceptors/ion channels and neuropeptides.

Histamine receptors on bovine peripheral blood lymphocytes.

Histamine receptors on bovine peripheral blood lymphocytes (PBL) were detected by three different methods: a rosetting technique, binding to histamine-bearing Sepharose beads and immunofluorescence staining. The rosetting technique used histamine-rabbit serum albumin (H-RSA) conjugated to bovine red blood cells to detect histamine receptors and this showed that 10.8% of bovine PBL were positive. A method using H-RSA conjugate coupled Sepharose beads also detected histamine receptor bearing PBL but was not quantitative. The indirect immunofluorescence method, by which the subpopulation of histamine receptor bearing lymphocytes can be easily double stained to concurrently identify the B cell marker, revealed that PBL, the B cell and T cell fraction of bovine PBL contained 18.4, 52.8 and 9.3% histamine receptor bearing cells, respectively. This method was found to be more stable and more easily quantifiable than the other two methods. Blocking tests using the histamine H1 receptor antagonist diphenhydramine and the histamine H2 receptor antagonist cimetidine suggested that bovine PBL have both H1 and H2 receptors on their surfaces. Addition of histamine into cultures of PBL at the concentration range 10(-6) to 10(-3) M suppressed the response of PBL to the mitogen phytohemagglutinin. The histamine induced suppression of mitogenesis could be reduced partially by the H2 receptor antagonist cimetidine, but not by the H1 antagonist diphenhydramine. It is possible that histamine induced suppression of PBL mitogenesis was mediated by H2 receptors on T cells.

Vaccines against arthropods.

The possibility of vaccinating hosts against blood-feeding arthropods using antigens derived from salivary gland, gut, and other tissues is reviewed. These vaccines directed against vector arthropods also have the potential to effect the arthropods capacity to transmit pathogens, and this is distinct from transmission-blocking vaccines that use antigens derived from pathogens. Antigen extracts have been used in attempts to vaccinate against fleas, lice, keds, flies, mosquitoes, and a number of tick species. A vaccine against the cattle tick, Boophilus microplus (Canestrini), using a recombinant antigen, has been tested under field conditions. Ticks feeding on vaccinated hosts are damaged by an immune response directed against their gut cells. Some die on the host, others engorge but their fecundity is reduced. The Commonwealth Scientific Industrial Research Organization-Biotechnology Australia tick vaccine against B. microplus is cited as a model for the development of other vaccines. It is suggested that the weaker effects of vaccines against insects as compared with ticks are related to the different structure and physiologies of the gut rather than being related to time spent on the vertebrate host. These differences in the effects of vaccines on insects may favor vaccines which block the passage of pathogens into vector insects. Vaccines against mosquitoes have been shown to reduce susceptibility of mosquitoes to arboviruses. The potential of the different vaccines is discussed.

Localization of a low abundance membrane protein (Bm86) on the gut cells of the cattle tick Boophilus microplus by immunogold labeling.

A preembedding immunogold technique was used to locate Bm86, an antigen from the gut digest cells of the cattle tick Boophilus microplus. Gut from partially engorged female ticks was everted to expose the cells, lightly fixed in 4% paraformaldehyde, and then incubated in rabbit antisera against a recombinant form of Bm86. Following incubation in a secondary antibody conjugated to 1-nm colloidal gold, Bm86 antigenic sites were visualized for both light and electron microscopy using silver enhancement. Bm86 was shown to be located predominantly on the microvilli of digest cells. Antiserum against a nonglycosylated Escherichia coli recombinant form of Bm86 was used to avoid cross-reactivity with carbohydrate epitopes of other digest cell proteins.

Effects of cattle tick (Boophilus microplus) infestation on the bovine immune system.

The immunosuppressive effect of experimental Boophilus microplus infestation on bovine peripheral blood lymphocytes (PBL) and on host antibody production to a protein antigen (ovalbumin) was examined. Boophilus microplus infestation caused a marginal decrease in the percentage of T lymphocytes in PBL, which was observed in both lightly (5000 larvae) and heavily (40,000 larvae) infested cattle, and began at the second infestation and continued until the end of the fourth infestation. The percentage of B lymphocytes in heavily tick-infested cattle was less than that in non-infested control cattle after the fourth infestation. The response of PBL from tick-infested cattle to phytohemagglutinin (PHA) was always less than that of tick-free cattle after the second infestation. No noteworthy differences were detected between the three stages of tick infestation, that is, 1 week before the peak of adult engorgement, the middle of the peak and 1 week after all ticks had dropped. Boophilus microplus saliva (100 microliters ml-1) suppressed 47% of the response of bovine PBL to PHA in vitro. This suppressive effect of saliva may contribute to the lower responsiveness of PBL from tick-infested cattle. Antibody production by tick-infested cattle was examined during the third and fourth heavy tick infestation. Tick-infested cattle showed a diminished response against ovalbumin after the second immunization. The immunosuppressive effects of tick infestation may play an important role in tick survival or in the transmission of tick-borne diseases in the field.

Native and baculovirus-expressed forms of the immuno-protective protein BM86 from Boophilus microplus are anchored to the cell membrane by a glycosyl-phosphatidyl inositol linkage.

A glycoprotein (BM86) from the gut cells of the cattle tick Boophilus microplus, when used to vaccinate cattle, has been shown to protect cattle from tick infestation. A recombinant BM86 protein is the principal component of a novel tick vaccine currently under development. The nature of the anchorage of BM86 to tick gut epithelial cells has been investigated using BM86 from B. microplus and recombinant BM86 proteins expressed in insect cells using the baculovirus expression system. BM86 from B. microplus and a full length recombinant BM86 are shown to be anchored to the extracellular surface of tick gut epithelial cells and baculovirus-infected insect cells, respectively by a glycosyl-phosphatidyl inositol membrane anchor. A recombinant BM86 truncated by the removal of a hydrophobic region coding for thirty amino acids at the carboxy-terminal end was secreted from baculovirus-infected Sf9 cells. This secreted form of recombinant BM86 showed strong protective activity against ticks in cattle vaccinated with this protein.

Successful vaccination against Boophilus microplus and Babesia bovis using recombinant antigens.

Current methods for the control of the cattle tick Boophilus microplus and the agent of bovine babesiosis, Babesia bovis are unsatisfactory. Effective immunological control of both parasites would have great advantages. However, naturally acquired immunity to the tick is generally unable to prevent serious production losses. A vaccine against the tick, based on a novel form of immunization, is being developed. A protective antigen has been isolated from the tick, characterized and produced as an effective, recombinant protein. A vaccine incorporating this antigen is currently undergoing field trials. In the Australian situation, improved tick control will probably increase endemic instability with respect to B. bovis. Fortunately, a trivalent, recombinant B. bovis vaccine has also been developed. This too is now undergoing pre-registration field trials.